Abstract
BackgroundNecroptosis is a type of regulated form of cell death that has been implicated in the pathogenesis of various diseases. Receptor-interacting protein 3 (RIP3), a member of the RIP family of proteins, has been reported as an important necroptotic pathway mediator in regulating a variety of human diseases, such as myocardial ischemia, inflammatory bowel disease, and ischemic brain injury. Our previous study showed that RIP3 was expressed in rat retinal ganglion cells (RGCs), where it was significantly upregulated during the early stage of acute high intraocular pressure. Furthermore, RIP3 expression was co-localized with propidium iodide (PI)-positive staining (necrotic cells). These results suggested that RIP3 up-regulation might be involved in the necrosis of injured RGCs. In this study, we aimed to reveal the possible involvement of RIP3 in oxygen glucose deprivation (OGD)-induced retinal ganglion cell-5 (RGC-5) necroptosis.MethodsRGC-5 cells were cultured in Dulbecco’s-modified essential medium and necroptosis was induced by 8 h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. RIP3 expression was detected by western blot and flow cytometry was used to detect the effect of RIP3 on RGC-5 necroptosis following OGD in rip3 knockdown cells. Malondialdehyde (MDA) lipid peroxidation assay was performed to determine the degree of oxidative stress.ResultsPI staining showed that necrosis was present in the early stage of OGD-induced RGC-5 cell death. The presence of RGC-5 necroptosis after OGD was detected by flow cytometry using necrostatin-1, a necroptosis inhibitor. Western blot demonstrated that RIP3 up-regulation may be involved in RGC-5 necroptosis. Flow cytometry revealed that the number of OGD-induced necrotic RGC-5 cells was reduced after rip3 knockdown. Furthermore, MDA levels in the normal RGC-5 cells were much higher than in the rip3-knockdown cells after OGD.ConclusionsOur findings suggest that RGC-5 cell necroptosis following OGD is mediated by a RIP3-induced increase in oxidative stress.
Highlights
Necroptosis is a type of regulated form of cell death that has been implicated in the pathogenesis of vari‐ ous diseases
The immunofluorescence results indicated that there was a large number of necrotic retinal ganglion cells (RGCs)-5 cells after 6 h reoxygenation following oxygen glucose deprivation (OGD); we chose this time point to analyze cellular necroptosis by flow cytometry with propidium iodide (PI)/Annexin V double staining following pretreatment with Nec-1 (RGC-5 cells were incubated with 10 μM Nec-1 for 24 h prior to OGD)
The results showed that necrosis occurred after OGD (Fig. 2b), but the number of necrotic (PI-positive) cells decreased significantly with Nec-1 pretreatment (Fig. 2c, d, P < 0.05)
Summary
Necroptosis is a type of regulated form of cell death that has been implicated in the pathogenesis of vari‐ ous diseases. Our previous study showed that RIP3 was expressed in rat retinal ganglion cells (RGCs), where it was significantly upregulated during the early stage of acute high intraocular pressure. RIP3 expression was co-localized with propidium iodide (PI)-positive staining (necrotic cells). These results suggested that RIP3 up-regulation might be involved in the necrosis of injured RGCs. In this study, we aimed to reveal the possible involvement of RIP3 in oxygen glucose deprivation (OGD)-induced retinal ganglion cell-5 (RGC-5) necroptosis. Several recent studies have suggested that receptor interacting protein 3 (RIP3) plays an important role in necroptosis in many cell types. Vieira et al [7] showed that RIP3 mediated neuronal cell death and its expression was upregulated in primary hippocampal neurons following OGD-induced injury. Dvoriantchikova et al [10] showed that mouse RGC necroptosis might be caused by inflammatory responses induced by RIP3
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